A New 4.0-Generation Dendrimer Phosphorescence Labeling Reagent and Its Application to Determination of Trace Alkaline Phosphatase by Affinity Adsorption Solid Substrate-room Temperature Phosphorimetry

A Triton X-100-4.0G-D （4.0G-D refers to a 4.0-generation dendrimer） was brought forward as a new phosphorescence labeling reagent. Two types of specific affinity adsorption （AA） reactions （direct method and sandwich method） were carried out between the labeling product of Triton X-100-4：0G-D-Wheat germ agglutinin （WGA） and alkaline phosphatase （ALP）, the product of AA reaction preserved the good characteristics of room temperature phosphorescence （RTP） of 4.0G-D and △Ip of the product was proportional to the content of ALP. According to the fact stated above, a new method for the determination of trace ALP by affinity adsorption solid substrate-room temperature phosphorimetry （AA-SS-RTP） was established on the basis of WGA labeled with the Triton X-100-4.0G-D. The detection limits were 0.20 ag·spot^-1 （corresponding concentration： 5.0×10^-16 g·mL^-1, namely 5.0×10^-18 mol·L^-1） for a direct method and 0.14 ag·spot^-1 （corresponding concentration： 3.5×10^-16 g·mL^-1, namely 3.5×10^-18 mol·L^-1） for a sandwich method, respectively. For their high sensitivity, good repeatability and high accuracy, the direct method and sandwich method have been successfully appfied to determine the content of ALP in human serum, and the results were coincided with the clinical detection results of the enzyme-linked immunosorbent assay method by the Zhangzhou Hospital of Traditional Chinese Medicine. Meanwhile, the mechanism for the determination of trace ALP by AA-SS-RTP was discussed.     

Fabrication of Pascal-triangle Lattice of Proteins by Inducing Ligand Strategy

A protein Pascal triangle has been constructed as new type of supramolecular architecture by using the inducing ligand strategy that we previously developed for protein assemblies. Although mathematical studies on this famous geometry have a long history, no work on such Pascal triangles fabricated from native proteins has been reported so far due to their structural complexity. In this work, by carefully tuning the specific interactions between the native protein building block WGA and the inducing ligand R-SL , a 2D Pascal-triangle lattice with three types of triangular voids has been assembled. Moreover, a 3D crystal structure was obtained based on the 2D Pascal triangles. The distinctive carbohydrate binding sites of WGA and the intralayer as well as interlayer dimerization of RhB was the key to facilitate nanofabrication in solution. This strategy may be applied to prepare and explore various sophisticated assemblies based on native proteins.     

Synthetic Mucin‐Like Glycopeptides as Versatile Tools to Measure Effects of Glycan Structure/Density/Position on the Interaction with Adhesion/Growth‐Regulatory Galectins in Arrays

Functional pairing of cellular glycoconjugates with tissue lectins is a highly selective process, whose determinative factors have not yet been fully delineated. Glycan structure and modes of presentation, that is, its position and density, can contribute to binding, as different members of a lectin family can regulate degrees of responsiveness to these factors. Using a peptide repeat sequence motif of the glycoprotein mucin‐1, the principle of introducing synthetic (glyco)peptides with distinct variations in these three parameters to an array‐based screening of tissue lectins is illustrated. Interaction profiles of seven adhesion/growth‐regulatory galectins cover the range from intense signals with core 2 pentasaccharides and core 1 binding for galectins‐3 and ‐5 to a lack of binding for galectin‐1 and also the galectin‐related protein, which was included as a negative control. Remarkably, the two tandem‐repeat‐type galectins‐4 and ‐8 were distinguished by core 1 sialylation, as the two separated domains were. These results encourage further synthetic elaboration of the glycopeptide library and testing of the network of natural galectins and rationally engineered variants of the lectins.     

Generation of a dynamic combinatorial library using sialic acid aldolase and in situ screening against wheat germ agglutinin

This paper describes the generation of a dynamic combinatorial library of sialic acid analogues using sialic acid aldolase. Addition of wheat germ agglutinin to the equilibrating libraries results in selective amplification of one or more members.     

Exploitation of phosphorescent labelling reagent of fullerol-fluorescein isothiocyanate and new method for the determination of trace alkaline phosphatase as well as forecast of human diseases

A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC)_n-DMA) was developed. The mode of action is based on the reactivity of the active -OH group in F-ol with the -COOH group of FITC to form an F-ol-(FITC)_n-DMA complex containing several FITC molecules. F-ol-(FITC)_n-DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC)_n-DMA (WGA and AP are wheat germ agglutinin and alkaline phosphatase, respectively) which improved the sensitivity using solid substrate room temperature phosphorimetry (SSRTP) detection. The proposed method provided high sensitivity and strong specificity for WGA-AP. The limit of detection (LD) was 0.15 ag AP spot⁻¹ for F-ol and 0.097 ag AP spot⁻¹ for FITC in F-ol-(FITC)_n-DMA, which was lower than the method using single luminescent molecules of F-ol-DMA and FITC-DMA to label WGA (0.20 ag AP spot⁻¹ for F-ol-DMA and 0.22 ag AP spot⁻¹ for FITC-DMA). Results for the determination of AP in human serum were in good agreement with those obtained by enzyme-linked immunosorbent assay. The mechanism of F-ol-(FITC)_n-DMA labelling of WGA was discussed.     

Dynamics ofLens culinaris agglutinin studied by red-edge excitation spectra and anisotropy measurements of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues

The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate bound toLens culinaris agglutinin and of the Trp residues of the protein was investigated. Red-edge excitation spectra and steady-state anisotropy as a function of temperature indicate that the TNS is bound rigidly. Red-edge excitation spectra, steady-state anisotropy as a function of sucrose and anisotropy decay experiments performed on Trp residues fluorescence prove that the internal fluorophore presents residual motion independent of the global rotation of the protein. Fluorescence anisotropy decay allows to calculate the rotational correlation time (351 ps) of this local motion. Quenching resolved emission anisotropy with iodide gives values equal to 0.257 and 0.112 for the anisotropies of the buried and the surface Trp residues, respectively. This result indicates that the Trp residues present at the surface of the protein have important local motions compared to those embedded in the protein matrix. The results obtained from TNS and Trp residues indicate that the agglutinin has different dynamic domains.     

Determination of glucose by affinity adsorption solid substrate-room temperature phosphorimetry based on concanavalin agglutinin labeled with fluorescein using 1.5-generation dendrimers as sensitizer

In the presence of the heavy atom perturber Pb(Ac)₂, fluorescein (HFin) can emit strong and stable room temperature phosphorescence (RTP) on the surface of a nitrocellulose membrane (NCM) at λ_ex ^max/λ_em ^max = 480/648 nm. It can be spiked with the 1.5-generation polyamidoamine dendrimers (abbreviated: D-1.5) to emit stronger RTP. It was found that a quantitative specific affinity adsorption (AA) reaction between concanavalin agglutinin (abbreviated: Con A) labeled with D-1.5-HFin and N-acetylglucosamine (G) could be carried out on the surface of NCM. The product of the reaction (D-1.5-HFin- Con A-G) could emit strong and stable RTP, and the ΔI_p was proportional to the content of G. According to the above facts, a new method for determination of G by affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) was established, based on Con A labeled with fluorescein using D-1.5 dendrimers molecules as sensitizer. The linear range of the sandwich method was 0.040–60 pg G spot⁻¹ (corresponding concentration range: 0.10–150 ng mL⁻¹; sample volume: 0.40 μL spot⁻¹). The regression equation of the working curve was ΔI_p = 6.880 + 5.610 m_G pg spot⁻¹, r = 0.9987. The working solutions containing 0.10 and 150 ng mL⁻¹ G were determined repeatedly 11 times, respectively. The corresponding RSDs were 2.9 and 3.8%. The detection limit of this method calculated by 3Sb/k was 0.021 pg spot⁻¹ (5.2 × 10⁻¹¹ g mL⁻¹). Compared with the direct method (detection limit: 0.078 pg spot⁻¹, linear range: 0.40–40 pg G spot⁻¹), the sensitivity was obviously improved and the linear range was wider. This method has been successfully applied to the determination of G in human plasma, as well as to the supervision and forecast of human diseases, for it is of good sensitivity, accuracy and precision. Correspondence: Jia-Ming Liu, Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou 363000, P.R. China     

Determination of trace alpha-fetoprotein variant by affinity adsorption solid substrate-room temperature phosphorimetry and its application to the forecast of human diseases

In the presence of ion perturber LiAc, 4-generation polyamidoamine dendrimers (4G-D) could emit strong and stable room temperature phosphorescence (RTP) signal at on nitrocellulose membrane (NCM), and Triton X-100 could sharply enhance the RTP signal of 4G-D. Triton X-100-4G-D was used to label concanavalin agglutinin (Con A) to get the labeling product Triton X-100-4G-D-Con A. Quantitative specific affinity adsorption (AA) reaction between Triton X-100-4G-D-Con A and α-fetoprotein variant (AFP-V) could be carried out on the surface of NCM, whose product Triton X-100-4G-D-Con A-AFP-V could emit strong and stable RTP and its ΔI_p was proportional to the content of AFP-V. According to the facts above, a new affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) for the determination of trace AFP-V by Con A labeled with Triton X-100-4G-D was established. Detection limits of this method were 0.23 fg spot⁻¹ (direct method, corresponding concentration: 5.8 × 10⁻¹³ g mL⁻¹) and 0.13 fg spot⁻¹ (sandwich method, corresponding concentration: 3.2 × 10⁻¹³ g mL⁻¹). It has been successfully applied to determine the content of AFP-V in human serum and forecast human diseases, for its high sensitivity, long RTP lifetime, good repeatability, high accuracy and little background perturbation with at the long wavelength area. Meanwhile, the mechanism for the determination of trace AFP-V using AA-SS-RTP was also discussed.     

Construction of Protein Probe with a His-tag and an Electron-transfer Peptide for a Target Protein Sensing

A protein probe with an electron-transfer peptide and a His-tag was designed to electrochemically sense a target protein. We selected tyrosine-rich (Y₄C) and tryptophan-rich (W₄C) peptides for use as electron-transfer agents. The peak for oxidation was based on the oxidations of the phenolic hydroxy groups in Y₄C and on the indole rings in W₄C. Asialofetuin (ASF) with galactose residues was the protein probe, and a galactose recognition protein, soybean agglutinin (SBA), was the target protein. A protein probe composed of an amino acid and carbohydrate residue was expected to be biocompatible. When voltammetric measurements were performed using a glassy carbon electrode, the oxidation peaks of H₆Y₄C and ASF-H₆Y₄C appeared at the same potential. The peak current of ASF-H₆Y₄C was 4-fold that of H₆Y₄C because of the stronger adsorption of ASF-H₆Y₄C onto the electrode. The electrode response of ASF-H₆Y₄C with SBA was half that of ASF-H₆Y₄C alone. By contrast, the peak current of ASF-Y₄CH₆ was higher than that of ASF-H₆Y₄C, which was the result of a greater degree of contact between the Y₄C moieties and an electrode. On the other hand, the voltammetric behaviors of ASF with W₄C and a His-tag were similar to those with Y₄C and a His-tag. The sensitivity of SBA using ASF-Y₄CH₆ was at the 10⁻¹³ M level. To confirm the function of the sensing system, measurements were performed in human serum with SBA and ASF-Y₄CH₆. When SBA was added, the serum had a concentration that ranged between 5.0×10⁻¹³ and 4.0×10⁻¹² M, and the amount of SBA that could be recovered ranged from 97 to 101%. Consequently, this system could be applied to the detection of SBA in serum.     

麦胚凝集素亲和色谱富集糖肽过程中的肽键裂解

蛋白质的糖基化是最重要的翻译后修饰之一,与蛋白质结构和功能的关系密切。凝集素亲和色谱是蛋白质糖基化研究中很常用的工具,不同的凝集素可以对不同的单糖或寡糖有特异的富集作用。麦胚凝集素（WGA）由于其特异作用的糖型广泛存在而成为使用最多的凝集素之一。在本研究中,发现将WGA用于糖肽亲和富集会导致部分肽段的降解,从而导致后续的肽段序列分析的失败。本文用4种标准蛋白质对这种现象进行了验证,结果表明肽段的降解可以发生在多个位点,其中较多地发生在酪氨酸、苯丙氨酸及亮氨酸的羧基端。这一结果提示：在糖蛋白质组研究中,如果应用WGA富集糖肽并采用质谱进行鉴定,则采用半酶切或非特异性酶切的检索策略更为合适。